Interpretive Summary: Because it takes two years to completed one generation of onion, classical crossing takes at least four years to classify cytoplasms as normal (N) male-fertile or male-sterile (S). Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time and expense required to classify onion cytoplasms. In this research, we sequenced the chloroplast DNAs of N and S-cytoplasmic onions, assembled and identified genes in the genomes, and identified single nucleotide polymorphisms (SNPs) distinguishing these cytoplasms. The sizes (153,538 and 153,355 basepairs) and gene orders were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small insertions or deletions in intergenic regions. Twenty-eight SNPs and two polymorphic restriction-enzyme sites distributed across 20 chloroplast genes were selected and validated using diverse onion populations previously classified as N or S-cytoplasmic using restriction fragment length polymorphisms. This research will be of interest to onion breeders in the public and private sectors by providing DNA differences useful for high throughput classification of onion cytoplasms as an aid to the development of hybrid-onion cultivars.

Technical Abstract:
Due the biennial generation time of onion, classical crossing takes at least four years to classify cytoplasms as normal (N) male-fertile or male-sterile (S). Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N and S-cytoplasmic onions, assembled and annotated the genomes, and identified single nucleotide polymorphisms (SNPs) distinguishing these cytoplasms. The sizes (153,538 and 153,355 basepairs), GC contents (36.8%), and gene orders were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions. Twenty-eight SNPs and two polymorphic restriction-enzyme sites distributed across 20 chloroplast genes were selected and validated using diverse onion populations previously classified as N or S-cytoplasmic using restriction fragment length polymorphisms. These chloroplast polymorphisms will be useful for high throughput classification of onion cytoplasms as an aid to the development of hybrid-onion cultivars.